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PubMed1994

Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers.

Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation assays with hepatic microsomes from rainbow trout (Oncorhynchus mykiss). Microsomes were prepared after 48 hr of treatment with beta-naphthoflavone (beta-NF), pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after ISF treatment than after beta-NF treatment for 7-methoxy-, 7-ethoxy-, 7-propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazone metabolism (about a 144-fold increase) compared with microsomes induced by beta-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the following order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pentoxy. When beta-NF-treated microsomes were used, the 7-alkoxyphenoxazones were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy = pentoxy, while the order of metabolism of the 7-alkoxycoumarins was: ethoxy >> butoxy > propoxy = methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxycoumarins. Treatment with beta-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy-, and butoxy- = pentoxy- = propoxyquinoline, in that order. ISF treatment significantly elevated the rate of metabolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. These results suggest that some of these new fluorescent substrates can be used to characterize induction of rainbow trout hepatic microsomal monooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of induction by beta-NF or other 3-methylcholanthrene-type P450 inducers. Distinction between ISF-type and beta-NF-type inducers in rainbow trout hepatic microsomes may best be made using 7-methoxycoumarin as a substrate. Distinction between ISF-type and DEX-type inducers and between beta-NF-type and DEX-type inducers may best be made using 7-methoxyphenoxazone as a substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Microsomal target proteins of metabolically activated aromatic hydrocarbons.

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.

Animals↗
PubMed1989

Microsomal metabolism of hexavalent chromium. Inhibitory effect of oxygen and involvement of cytochrome P-450.

The reduction of hexavalent chromium (Cr(VI] by rat liver microsomes was studied. With 15-120 microM Na2CrO4 microsomes (0.5 mg protein/ml) effectively reduced Cr(VI) in the presence of NADPH provided anaerobic conditions. Phenobarbital (PB) and Aroclor 1254 (PCB) pretreatment increased microsomal Cr(VI) reduction while CoCl2 reduced the rate. The rates with 30 microM Na2CrO4 were: 6.4 +/- 0.1, 7.8 +/- 0.7, 13.4 +/- 0.5, 2.95 +/- 0.09 nmol Cr.mg prot.-1 min-1 for control, PB, PCB and cobalt pretreated microsomes respectively. Kinetic studies gave a Michaeli-Menten like first-order kinetics with increases both in Km and Vmax values after pretreatment with PB or PCB. CO partly inhibited the microsomal Cr(VI) reduction. The CO-sensitive reduction rate was directly correlated to the cyt. P-450 content of the different microsomal preparations. Substituting NADH for NADPH gave approximately 27% lower activity with 30 microM Na2CrO4. This activity was neither inducible by cyt. P-450 inducers nor influenced by CO. Oxygen 1.0% and 0.10% gave approximately 100% and 30% inhibition of Cr(VI) reduction (30 microM Na2CrO4) respectively, and an uncompetitive like inhibitory pattern was found. No redox cycling of Cr(VI) was seen. 51Cr binding to the microsomes was approximately 10% after complete reduction of 30 microM Na2CrO4. Externally added FMN, Fe3+-ADP and nitrobenzen stimulated microsomal Cr(VI) reduction. A 60% higher reduction rate of Cr(VI) by isolated hepatocytes was found during anaerobic in comparison with aerobic conditions.

Animals↗
PubMed1990

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol.

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.

Animals↗
PubMed1984

Augmentation of cholesterol esterification in the microsomal membrane by the removal of membrane-bound ribosomes.

Acyl-CoA: cholesterol O-acyltransferase (ACAT) activity in rough microsomes was enhanced (2-fold) by the removal (40%) of ribosomes from the microsomal membrane with RNase. Although EDTA was as efficient as RNase in the removal of ribosomes the stimulation (3.2-fold) of ACAT activity was even more, suggesting that additional effects were induced by EDTA. Reconstitution of EDTA-treated microsomes with ribosomes decreased the cholesterol-esterifying activity (40%) of the degranulated microsomes. Alternate possibilities were considered for the enhancement of ACAT activity by EDTA, namely, suppression of the hydrolysis of added palmitoyl-CoA substrate, the hydrolysis of cholesteryl ester, and the removal of metal suppressor of ACAT activity. Neither acyl-CoA hydrolase nor cholesteryl ester hydrolase activity was decreased after degranulation of microsomes. ACAT activity of EDTA-treated microsomes compared to the control was enhanced rather than suppressed after the addition of Ca2+, Mg2+, and Ba2+ ions whereas other metal ions (Co2+, Cu2+, Zn2+) almost completely suppressed ACAT activity in both the EDTA-treated and buffer-treated microsomes. It is concluded that the enhanced ACAT activity was largely due to the removal of ribosomes from the microsomal membrane.

Animals↗
PubMed1977

Studies of the mechanism of augmented synthesis of cholesteryl ester in atherosclerotic rabbit aortic microsomes.

A study was undertaken to test the hypothesis that an abnormally high concentration of acyl-CoA:cholesterol acyltransferase in atherosclerotic microsomes is partly responsible for augmented esterification of cholesterol. We approached the problem indirectly by measuring the incorporation of radioactivity into cholesteryl ester from [1-14C]palmityl-CoA in normal microsomes after enrichment of their concentration of microsomal free cholesterol to levels characteristic of atherosclerotic microsomes. Elevation of free cholesterol content induced increased cholesterol esterification approximately linearly over the range studied. The cholesterol-esterifying activity of atherosclerotic microsomes was not greater than that of normal microsomes having the same concentration of cholesterol. The results suggest that, with acyl-CoA constant, augmented cholesterol esterification in atherosclerotic microsomes is an effect of high microsomal cholesterol concentrations and not due to an increase in the concentration of the enzyme.

Acyl Coenzyme A↗
PubMed1985

Some characteristics of human adrenal microsomal 21-hydroxylase activity.

The following general characteristics of 21-hydroxylase activity were determined using pooled microsomes obtained from three glands. Enzyme activity exhibited a broad pH dependence, being optimal between pH 7.4-pH 7.8, and was maximal with NADPH in the range 2 to 4.75 X 10(-4)mol/l. No microsomal 21-hydroxylase activity was detected in the absence of NADPH or substrate and when heat denatured microsomes were employed. Enzyme activity was depressed by greater than 75% in the presence of 100% oxygen or nitrogen. In a second set of experiments, microsomal fractions were prepared individually from 7 glands. In the presence of 17 alpha-hydroxy progesterone (2.0 X 10(-7) and 2.0 X 10(-6)mol/l) product formation was linear with time for up to 90 s when the microsomal protein concentration was 5, 10 and 20 micrograms/ml. Between 5 and 30% of the substrate was converted during the first 60 s. In 5/7 of the glands the addition of the autologous cytosol (20 micrograms protein/ml) was without effect, and enzyme activity (using a 60 s reaction and either 2.0 X 10(-7) or 2 X 10(-6)mol/l 17 alpha-hydroxy progesterone was directly proportional to the microsomal protein concentration (range 0-20 micrograms/ml). With the other 2 adrenals 21-hydroxylation was not proportional to the same range of microsomal protein concentrations, although it became so upon the addition of cytosol, which significantly augmented activity. There was considerable variation in enzyme activity between glands from different individuals (Vmax ranging from 2.6 to 16.6 X 10(-9) mol/min/mg protein) and in the apparent Km's (from 0.22 to 1.1 X 10(-6)mol/l). In the two preparations sensitive to cytosol, the Vmax increased 2-fold, and the Km was 3 times lower. Cytosol was without effect upon the kinetic characteristics of the other 5 microsomal preparations. Ascorbic acid (1 X 10(-3) mol/l) depressed enzyme activity by 25-43% whereas oxidised and reduced glutathione (1 X 10(-3) mol/l) showed a slight and variable effect upon 21-hydroxylation.

17-alpha-Hydroxyprogesterone↗
PubMed1993

Phospholipid requirement of epididymal testosterone 5 alpha-reductase and phospholipid composition of epididymal microsomes.

We have investigated phospholipid requirement for testosterone 5 alpha-reductase solubilized from microsomal and nuclear fractions of rat epididymis. The 5 alpha-reductase from microsomal fraction was stimulated by phosphatidylcholine (PC) with long acyl-chain lengths, but inhibited by short chain PC. The nuclear enzyme activity was weakly activated by PC with various acyl-chain lengths tested. Synthetic phosphatidylserine (PS), such as dioleoylPS, most strongly stimulated the microsomal enzyme activity, but did not exhibit any activation of the nuclear enzyme activity. Endogenous phospholipids, such as PC, PS, and phosphatidylethanolamine (PE) separated from bovine epididymal microsomes were tested for their stimulatory effects on microsomal and nuclear enzymes. Among these endogenous phospholipids, PS most greatly stimulated the microsomal 5 alpha-reductase activity, whereas both PC and PE weakly activated the enzyme activity. On the other hand, endogenous PC and PS had no ability to support the nuclear enzyme activity. The fatty acid compositions of PC and PS from bovine epididymal microsomes were determined, in order to elucidate the relationship between 5 alpha-reductase activation by these phospholipids and the structure of their acyl chains. The relative content of fatty acids in PC, in a decreasing order, was palmitate > linoleate > oleate; that in PS was stearate > oleate > palmitate. Based on these observations, the roles of microsomal PS and PC in epididymal 5 alpha-reductase reaction will be discussed.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1991

The arylation of microsomal membrane proteins by acetaminophen is associated with the release of a 44 kDa acetaminophen-binding mouse liver protein complex into the cytosol.

When analyzed by Western blotting with affinity purified antibodies against acetaminophen, proteins of molecular weight 44 and 58 kDa appear to be the major macromolecular targets in livers of mice administered hepatotoxic concentrations of acetaminophen. In this study, we have examined the characteristics and biochemical properties of the 44 kDa acetaminophen-binding protein in mouse liver. Data are presented which indicate that the 44-kDa protein is the earliest detectable protein targeted by acetaminophen; 30 min after acetaminophen administration in vivo, the binding to the 44 kDa protein is primarily localized in the microsomal fraction. After 1 hr, the 44 kDa acetaminophen-binding protein can be detected in both the microsomes and the cytosol. Extractions of microsomes with Triton X-114 or 1 M NaCl suggests that the acetaminophen-bound 44-kDa protein behaves as a peripheral membrane protein associated with the endoplasmic reticulum by ionic interactions. The cytosolic and microsomal 44-kDa proteins possess similar biochemical properties; both exist natively as components of a protein complex of greater than 200 kDa and both consist of two major isovariants with isoelectric points of 7.0 and 7.1 on two-dimensional gels. When N-acetyl-p-benzoquinone imine, the reactive metabolite of acetaminophen, is incubated with cytosolic or microsomal fractions from control liver, targeting of a 44-kDa protein is only observed in the microsomes. However, when acetaminophen is activated in an NADPH-regenerating microsomal system in vitro, some of the microsomal 44-kDa protein complex can be solubilized and released into the cytosol. Thus, acetaminophen administration can alter the subcellular distribution of at least one protein target in the cell.

Acetaminophen↗
PubMed1991

Epoxidation of 4-vinylcyclohexene by human hepatic microsomes.

Carcinogenicity studies have shown that chronic administration of 4-vinylcyclohexene (VCH) will induce ovarian tumors in B6C3F1 mice but not F-344 rats. This occurs because the blood level of the ovotoxic VCH metabolite, VCH-1,2-epoxide, is dramatically higher in VCH-treated female mice compared with rats. This species difference in VCH epoxidation is also reflected in the rate of VCH metabolism by hepatic microsomes (female mouse greater than female rat). The present study assessed the ability of microsomes obtained from human liver to metabolize VCH to epoxides since humans are exposed to VCH in certain occupational settings. The production of VCH-1,2-epoxide and VCH-7,8-epoxide from VCH (1 mM) by human hepatic microsomes was linear with respect to microsomal protein concentration (0.25-1.0 mg/ml) and incubation time (5-20 min). VCH-1,2-epoxide was the major metabolite, while the rate VCH-7,8-epoxide formation was about 6-fold lower and in some cases was below the limit of detection. There was no dramatic difference in the rate of VCH epoxidation by hepatic microsomes obtained from male and female humans. The rate of VCH-1,2-epoxide formation by female human hepatic microsomes was 0.71 +/- 0.35 nmol/mg microsomal protein/min (n = 4). This is 13- and 2-fold lower than the rate of VCH-1,2-epoxide formation by female mouse and rat hepatic microsomes, respectively. Therefore, if the rate of hepatic VCH epoxidation is the main factor which determines the ovotoxicity of VCH, then the results of these studies suggest that rats are the more appropriate animal model for extrapolation of animal data to humans.

Animals↗
PubMed1992

Rat liver microsomal UDP-glucuronosyltransferase activity toward thyroxine: characterization, induction, and form specificity.

Glucuronidation of thyroxine (T4) by liver microsomal UDP-glucuronosyltransferase (UDP-GT) is a predominant pathway by which T4 is deactivated. This study was conducted to characterize in vitro T4 UDP-GT activity in rat liver microsomal preparations, to determine if T4 glucuronidation is mediated by a particular form of UDP-GT, and to determine if T4 glucuronidation can be increased by microsomal enzyme inducers. Characterization of microsomal T4 UDP-GT activity led to the establishment of optimal assay conditions. UDP-GT activity toward T4 was determined in hepatic microsomal preparations from Wistar and Gunn rats, a mutant strain of Wistar rats deficient in several forms of UDP-GT. Hepatic microsomal preparations from Gunn rats glucuronidated T4 at one-third the rate catalyzed by microsomal preparations from Wistar rats. To determine the effect of four inducers that each increase a separate class of UDP-GT, phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16 alpha-carbonitrile (PCN), clofibrate (CLO), saline, or corn oil was administered to male Sprague-Dawley rats ip for 4 days. T4 UDP-GT activity was increased by PB, 3MC, PCN, and CLO 88, 150, 100, and 160%, respectively on a per-milligram-microsomal-protein basis and 138, 125, 100, and 145% on a per-kilogram-body-weight basis, respectively. Therefore, all four classes of UDP-GT inducers increase T4 glucuronidation, suggesting that T4 is not a selective substrate for a particular form of UDP-GT.

Animals↗
PubMed1989

Interspecies homology of cytochrome P-450: toxicological significance of cytochrome P-450 cross-reactive with anti-rat P-448-H antibodies in liver microsomes from dogs, monkeys and humans.

The mutagenic activation of various promutagens by liver microsomes from dogs, monkeys and humans was investigated. Dog liver microsomes efficiently catalyzed the mutagenic activation of Trp-P-2 and Glu-P-1 followed by IQ and AAF. Monkey liver microsomes were most active in the activation of IQ followed by Glu-P-1, AAF and Trp-P-2. Although there were remarkable individual differences, human liver microsomes were found to be most active in the mutagenic activation of IQ followed by Trp-P-2, Glu-P-1 and AAF. Antibodies against rat P-448-H inhibited the mutagenic activation of Glu-P-1, Trp-P-2 and IQ in rat and dog liver microsomes, and Glu-P-1 and Trp-P-2 in monkey liver microsomes. The activation of Glu-P-1 and IQ in human liver microsomes was also strongly inhibited by anti-P-448-H antibodies. The amounts of cytochrome P-450 cross-reactive with anti-P-448-H antibodies in human liver microsomes highly correlated with the capacity to activate Glu-P-1, Trp-P-2 and IQ but not AAF.

2-Acetylaminofluorene↗
PubMed1994

Functional and structural membrane topology of rat liver microsomal glutathione transferase.

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11-35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2'-dithiobis(5-nitrobenzoic acid)) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42-154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1988

Comparative metabolism of phenanthrene and benzo[f]quinoline by rat liver microsomes.

The metabolism of benzo[f]quinoline (BfQ) and its carbon analog phenanthrene has been compared in incubations with liver microsomes from control, 3-methylcholanthrene (3-MC)- and phenobarbital (PB)-pretreated rats. The rates of phenanthrene metabolism by the three types of microsomes were 0.7, 4.1 and 1.5 nmol/mg protein per min, respectively; the values for BfQ were 0.5, 3.7 and 2.5, respectively. Besides N-oxidation, the metabolism of BfQ by all the above microsomes was almost exclusively at the benzo-ring (49-69%) while that of phenanthrene was predominantly at the K-region (50-71%). Phenanthrene-1,2-dihydrodiol, a precursor of the bay-region diol epoxide of phenanthrene, was produced many times more than phenanthrene-3,4-dihydrodiol by both 3-MC- and PB-induced microsomes. While BfQ-7,8-dihydrodiol, the precursor of the bay-region diol epoxide of BfQ, was the predominant metabolite with 3-MC-induced microsomes, it was a minor metabolite with PB-induced microsomes. The benzo-ring oxidation of BfQ, but not of phenanthrene, was position-specific, i.e. predominantly 7,8-oxidation by 3-MC-induced microsomes and 9,10-oxidation by PB-induced microsomes, and implies that aza-substitution results in a site-specific attack by different cytochromes P-450.

Animals↗
PubMed1983

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation.

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O2 consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radicals. This protein may be an important defense against free radical injury to the microsomal membrane.

Animals↗
PubMed1991

Microsomal chitinase activity from Candida albicans.

Chitinase (E.C. 3.2.1.14) was characterized in microsomal fractions from yeast cells of Candida albicans. Following six washes with buffer (50 mM Bis-Tris.Cl, pH 6.5), enzyme activity of microsomes fell markedly to 0.3% of total and 6% of the specific activity detected in the low-speed supernatant (9000 X g) of a cell lysate. An apparently zymogenic, microsomal chitinase activity became more readily detectable with washing and after six washes enzyme activity was activated 1.7-fold following pre-incubation with trypsin. The following properties of microsomal chitinase were closely comparable with those for cytosolic chitinase (indicated in parentheses): Km = 2.1 mg chitin per ml (2.9 mg chitin per ml); temperature optimum = 45 degrees C (45 degrees C); inhibition by allosamidin competitive, Ki = 0.29 microM (competitive, Ki = 0.23 microM). A range of detergents solubilized and activated microsomal chitinase in a highly specific manner. Following density gradient centrifugation of microsomes, chitinase was distributed approximately evenly throughout the gradient suggesting that microsomal chitinase is not associated exclusively with any one membrane component. The possible morphogenetic role of microsomal chitinase is discussed in relation to the potential of this enzyme as a target for highly specific antifungal agents.

Acetylglucosamine↗
PubMed1996

Role of cytochrome P-450 in the stimulation of microsomal production of reactive oxygen species by ferritin.

Microsomes can remove iron from ferritin by a superoxide-dependent reaction. The released iron can then catalyse formation of a variety of reactive oxygen species. Experiments were carried out to evaluate the role of cytochrome P-450 in the release of iron from ferritin, and whether induction of certain P-450 isoforms alters ferritin-dependent reactive oxygen radical production. Rats were treated with phenobarbital, 3-methylcholanthrene, 4-methylpyrazole, or saline to produce microsomes with varying P-450 content and composition. Oxidation of 2,7'-dichlorofluorescein diacetate to a fluorescent product and chemiluminescence were used as indices of production of reactive oxygen species. The extreme sensitivity of these reactions to trolox, a potent chain-breaking oxidant, indicates the involvement of lipid peroxidation products in these reactions. In the absence of ferritin, formation of reactive oxygen species was higher in microsomes from the treated rats compared to saline controls when results were expressed on a per mg protein basis but not per nmol P-450, suggesting that the increased content of total P-450 (2-fold increases) rather than the population of isoforms was responsible for the increase. Superoxide dismutase had no effect on the non-ferritin catalyzed reactions. Ferritin increased production of reactive oxygen species with all the microsomal preparations; the increase by ferritin was completely prevented by superoxide dismutase. The net increase by ferritin was higher in microsomes from the treated rats compared to saline controls, but this, again, largely reflected the increased content, rather than the type of isoforms of P-450 present. Similar results were obtained with either NADPH or NADH as microsomal reductants, although NADPH was much more effective in supporting ferritin-dependent reactive oxygen formation. In microsomes from phenobarbital-treated rats, anti-CYP2B1/B2 IgG completely prevented the NADPH- and NADH-dependent increases in reactive oxygen formation produced by ferritin. Anti-cytochrome b5 IgG produced partial inhibition of the ferritin-stimulation. These results indicate that P-450, and to a lesser extent, cytochrome b5, play a role in the ferritin-dependent increase in formation of reactive oxygen species with either NADPH or NADH, most likely reflecting the requirement of these enzymes for microsomal production of superoxide anion.

Animals↗
PubMed1986

Lipid composition of liver microsomes from rats treated with acetone.

The metabolism of certain compounds by rat hepatic microsomal preparations obtained from acetone-treated rats is greater than that measured in microsomes obtained from control rats. Since the lipid composition of membranes can significantly influence metabolism catalyzed by membrane-bound enzymes, studies were conducted in order to determine whether acetone-induced alterations in microsomal metabolism are associated with changes in the lipid composition of these membranes. Phospholipid and cholesterol concentrations were measured in extracts of rat liver microsomes obtained from rats treated with acetone, sodium phenobarbital, or vehicle. Acetone treatment did not alter the phospholipid and cholesterol content of the microsomes, whereas sodium phenobarbital (positive control) caused a significant decrease in cholesterol content/mg microsomal protein. These data indicate that acetone-induced alterations in microsomal metabolism may not be attributed to changes in the phospholipid and cholesterol content of the microsomes.

Acetone↗